WO2022219967A1 - Copolymère, kit de préparation de conjugué anticorps-copolymère, conjugué anticorps-copolymère, procédé de concentration d'antigène et procédé de détection d'antigène - Google Patents

Copolymère, kit de préparation de conjugué anticorps-copolymère, conjugué anticorps-copolymère, procédé de concentration d'antigène et procédé de détection d'antigène Download PDF

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WO2022219967A1
WO2022219967A1 PCT/JP2022/009632 JP2022009632W WO2022219967A1 WO 2022219967 A1 WO2022219967 A1 WO 2022219967A1 JP 2022009632 W JP2022009632 W JP 2022009632W WO 2022219967 A1 WO2022219967 A1 WO 2022219967A1
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antibody
copolymer
group
formula
antigen
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Japanese (ja)
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充宏 荏原
アーメッドナビル アーメッドエルセイト トルバ
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国立研究開発法人物質・材料研究機構
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Priority to EP22787895.6A priority Critical patent/EP4324858A1/fr
Priority to JP2023514514A priority patent/JPWO2022219967A1/ja
Priority to US18/272,097 priority patent/US20240168017A1/en
Publication of WO2022219967A1 publication Critical patent/WO2022219967A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/56—Acrylamide; Methacrylamide
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/60—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/14—Esterification
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the present invention relates to a copolymer, an antibody-copolymer conjugate preparation kit, an antibody-copolymer conjugate, a method for concentrating an antigen, and a method for detecting an antigen.
  • Temperature-responsive polymers are known that undergo a phase transition in response to changes in the temperature of the solution and change their physical properties.
  • Patent Document 1 describes a polymer of "an isopropylacrylamide derivative having an azide group or an alkyne group.”
  • an object of the present invention is to provide a copolymer that can be used to concentrate antigens by immobilizing antibodies. Another object of the present invention is to provide an antibody-copolymer conjugate preparation kit, an antibody-copolymer conjugate, a method for concentrating an antigen, and a method for detecting an antigen.
  • [1] A copolymer containing unit 1 described below and unit 2 described below.
  • [2] The copolymer according to [1], wherein the content of the unit 2 is 1.0 to 30.0 mol% based on 100 mol% of all repeating units.
  • [3] The copolymer according to [1] or [2], wherein the unit 2 is a unit 3 represented by formula 3 described later or a unit 4 represented by formula 4 described later.
  • [4] The copolymer according to any one of [1] to [3], further comprising a repeating unit represented by formula 5 described below.
  • [5] The copolymer according to any one of [1] to [4], wherein the content of the unit 2 is 5 to 30 mol% based on 100 mol% of all repeating units.
  • a linker represented by Formula 4 described later and the antibody are bound via an amide bond derived from an amino group of the antibody to prepare an antibody-linker conjugate; preparing a mixture B containing an antibody-linker conjugate, a unit 1 described later, and a copolymer containing a unit 2 described later; and the alkynylene group of the copolymer by reacting the azide group and the alkynylene group of the copolymer to form the antibody-copolymer conjugate.
  • a copolymer that can be used for antigen concentration can be provided by immobilizing antibodies.
  • the present invention can also provide an antibody-copolymer conjugate preparation kit, an antibody-copolymer conjugate, a method for concentrating an antigen, and a method for detecting an antigen.
  • FIG. 4 shows the evaluation results of click reactions between antibodies and different concentrations of synthetic polymers by SDS-PAGE.
  • FIG. 4 depicts enrichment of COVID-19 recombinant protein with varying concentrations of antibody conjugate.
  • LFIA concentration effect of COVID-19 antibody temperature responsive polymer by lateral flow immunoassay
  • the copolymer according to the embodiment of the present invention includes a repeating unit (unit 1) represented by the following formula 1 and a repeating unit represented by the following formula 2 It is a copolymer containing (unit 2).
  • Unit 1 shows a temperature response of 32° C. in LCST (Lower Critical Solution Temperature) of a polymer constituted by itself to water. By including unit 1, the specific copolymer has temperature responsiveness.
  • LCST Lower Critical Solution Temperature
  • the content of the unit 1 contained in the specific copolymer is not particularly limited, and is typically preferably 1 to 99 mol% when the total repeating units are 100 mol%.
  • the specific copolymer has a more sensitive temperature responsiveness, and / or the LCST is easy to control in a temperature range suitable for antigen concentration (specifically, the LCST is room temperature to 40 ° C. from the point of view, the content of unit 1 in the specific copolymer is preferably more than 50 mol%, more preferably 60 mol% or more, preferably 97 mol% or less, and more preferably 90 mol% or less. preferable.
  • X 1 represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, and from the viewpoint that the specific copolymer has more sensitive temperature responsiveness, A hydrogen atom or a linear alkyl group having 1 to 4 carbon atoms is preferable, and a hydrogen atom or a methyl group is more preferable.
  • unit 1 is preferably a unit based on a monomer (monomer) represented by formula 1' below.
  • X 1 has the same definition as X 1 in Formula 1, and the preferred forms are also the same.
  • the monomer represented by Formula 1' may be synthesized, for example, by a known method, for example, the method described in Examples below, or may be a commercially available product.
  • the specific copolymer has units 2 represented by Formula 2.
  • Unit 2 has one site (click reaction site) containing a cyclic alkyne (alkynylene group) per repeating unit, and is combined with an azide group of a linker described later by click reaction to easily form a complex. can form.
  • a linker and a protein such as an antibody
  • the protein can be fixed to the specific copolymer via the linker.
  • antibody-copolymer conjugates can be made.
  • the produced conjugate causes aggregation and precipitation at a temperature of LCST or higher due to the temperature responsiveness derived from the specific copolymer. This can be used to concentrate antigens corresponding to antibodies.
  • the content of the unit 2 in the specific copolymer is not particularly limited, but is typically preferably 1 to 99 mol% when the total repeating units of the specific copolymer are taken as 100 mol%.
  • it is preferably 1 mol% or more, more preferably 2 mol% or more, further preferably 5 mol% or more, and 30 mol % or less is preferable, and 20 mol % or less is more preferable.
  • Unit 2 is typically more hydrophobic than other repeating units, and increasing the content of unit 2 can lower the LCST. In addition, the higher the content of Unit 2, the easier the introduction of the antibody.
  • the content of unit 2 is preferably 1 mol% or more, more preferably 2 mol% or more, further preferably 5 mol% or more, and 10 mol %, preferably 30 mol % or less, more preferably 20 mol % or less.
  • X 2 represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, and from the viewpoint that the specific copolymer has more sensitive temperature responsiveness, A hydrogen atom or a linear alkyl group having 1 to 4 carbon atoms is preferable, and a hydrogen atom or a methyl group is more preferable.
  • L2 represents a divalent group.
  • L 2 is not particularly limited, but -O-, -S-, -C(O)-, -C(O)O-, -OC(O)O-, -NR A - (R A is a hydrogen atom or a monovalent substituent), a linear, branched or cyclic aliphatic hydrocarbon group having 1 to 20 carbon atoms, a monocyclic or condensed ring having 6 to 20 carbon atoms and at least one selected from the group consisting of a combination of these aromatic hydrocarbon groups.
  • —O—, —C(O)—, —NR A — a straight-chain alkylene group having 1 to 10 carbon atoms, from the viewpoint of obtaining a copolymer having a more excellent effect of the present invention , and more preferably one group selected from the group consisting of a combination thereof, and at least one group selected from the group consisting of -O-, -C(O)-, and -NR A - is more preferred, and -0-, -C(O)-, or -NH- is particularly preferred.
  • R 1 is a hydrogen atom, a halogen atom, —OR 5 , —NO 2 , —CN, —S(O) 2 R 5 , an alkyl group having 1 to 24 carbon atoms, and 2 to 2 carbon atoms.
  • R 1 selected from the group consisting of 24 alkenyl groups and (hetero)aryl groups having 6 to 24 carbon atoms, two or more R 1 may be bonded together to form a ring
  • R 5 is selected from the group consisting of hydrogen, a halogen atom, an alkyl group having 1 to 24 carbon atoms, and a (hetero)aryl group having 6 to 24 carbon atoms
  • Z is C(R 1 ) 2 , O, S and NR 1
  • a′ is an integer from 0 to 8
  • a′′ is an integer from 0 to 8
  • the sum of a′ and a′′ is less than 10.
  • a plurality of R 1 may be the same or different.
  • Unit 2 preferably has, for example, a structure (click reaction site) represented by formula C below, where "*" represents the binding site with L2.
  • a structure represented by the following formula C as a site (monovalent group) bonded at the position of "*" after the wavy line represented by the following formula.
  • Unit 2 is preferably at least one selected from the group consisting of units represented by the following formulas, from the viewpoint of obtaining a copolymer having more excellent effects of the present invention.
  • L21 represents a divalent group, has the same meaning as L2 in formula 2 , and the same applies to preferred forms.
  • X2 has the same definition as X2 in formula 2 , and the preferred forms are also the same.
  • unit 2 is preferably unit 3 or unit 4 represented by the following formula 3 or 4.
  • X 2 represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms
  • L 3 represents -O-, -S- and -NR B -, wherein R B represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and n is an integer of 1 to 10 and in Formula 4, L 2 represents a divalent group, and the preferred form is the same as the divalent group for L 2 in Formula 2.
  • the method for synthesizing unit 2 is not particularly limited, and known synthesis methods can be used. However, since unit 2 can be obtained more easily, a precursor compound is used for unit 2' represented by the following formula 2' to give unit 2 is preferred.
  • R C is a reactive substituent, and specific examples thereof include a hydroxy group, an amino group, a carboxy group, a glycidyl group, a mercapto group, a hydroxysuccinimide ester, and a maleimide. preferable.
  • the precursor compound for example, a compound having a click reaction site and a group capable of reacting with the above RC may be used, and a commercially available product may be used, or a compound synthesized by a known method may be used. .
  • the specific copolymer can be synthesized more easily by using a precursor compound having a structure (click reaction site) represented by the formula C and a carboxy group.
  • the click reaction site is difluorinated cyclooctyne
  • the method for synthesizing the precursor compound is described in J. Am. Am. Chem. Soc. 2008, 130, 34, 11486-11493, schemes 1 and 2 can be used.
  • the click reaction site is dibenzazacyclooctyne
  • the method described in Scheme 1 of Chemical Communications (2010), 46(1), 97-99 can be used.
  • BCN bis(bicyclo[6.1.0]nonyne)
  • DBCO DBCO
  • a substituent such as an amino group, a hydroxy group, and a carboxy group are added
  • maleimide, and , and hydroxysuccinimide-esterified products are commercially available, and these can also be used as precursor compounds.
  • the unit 2' is preferably the unit 5 represented by the following formula 5 in that the unit 2 can be obtained more easily.
  • a precursor compound having a carboxy group and a structure represented by formula C may be used as the precursor compound.
  • the LCST when synthesizing the specific copolymer, if there is an unreacted unit 5, in other words, if the specific copolymer contains units 1, 2, and 5, the LCST is adjusted to the high temperature side. can do. Since unit 5 is highly hydrophilic compared to other repeating units, the LCST can be adjusted to the high temperature side by increasing the content of unit 5.
  • X 5 represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, and from the viewpoint that the copolymer has more sensitive temperature responsiveness, A hydrogen atom or a linear alkyl group having 1 to 4 carbon atoms is preferable, and a hydrogen atom or a methyl group is more preferable.
  • the content of the unit 5 in the specific copolymer is not particularly limited, it is preferably 0 to 15 mol% when the total repeating units of the specific copolymer are 100 mol%.
  • the number average molecular weight of the specific copolymer is typically preferably from 2,000 to 100,000, more preferably from 5,000 to 50,000, and even more preferably from 10,000 to 30,000.
  • the number average molecular weight is 5000 or more, it is easy to obtain better concentration efficiency (in other words, it is easy to obtain better temperature responsiveness). It is easy to form, or the antigen-antibody reaction is more likely to proceed.
  • the number average molecular weight is preferably 15,000 or more, preferably 20,000 or more, in that more excellent concentration efficiency can be obtained when an antibody-copolymer conjugate prepared from the obtained copolymer is used. It is more preferable to have In general, antibodies are often highly hydrophilic, and in such cases, a copolymer having better concentration efficiency is required. , it is easy to obtain excellent concentration efficiency.
  • the ratio of the molecular weight of the specific copolymer to the molecular weight of the antibody (specific copolymer/antibody) is not particularly limited, but is preferably 1/30 to 1/5.
  • the molecular weight dispersity (PDI) of the specific copolymer is not particularly limited, it is preferably 1.5 or less, more preferably 1.3 or less, from the viewpoint of obtaining a copolymer having a more excellent effect of the present invention. .
  • the method for producing the specific copolymer is not particularly limited, but it is preferable to have the following step (1) and step (2) in this order from the viewpoint that the specific copolymer can be produced more easily.
  • Step (1) A step of copolymerizing the monomers represented by the following formulas 1' and 3' to obtain a copolymer (precursor).
  • X 1 and X 2 are respectively synonymous with X 1 in formula 1 and X 2 in formula 2′, and preferred embodiments are also the same. .
  • the method of copolymerizing the above monomers is not particularly limited, and it is preferable to use a living polymerization method such as a living radical polymerization method, a living anion polymerization method, and a living cationic polymerization method.
  • a living polymerization method such as a living radical polymerization method, a living anion polymerization method, and a living cationic polymerization method.
  • the living radical polymerization method is preferable from the viewpoint that the copolymer (precursor) can be obtained more easily.
  • the living radical polymerization method is based on establishing a rapid equilibrium between a small amount of growing radical (free radical) species and a large amount of dormant (dormant) species in the growth reaction by applying heat, light, metal catalyst, etc. there is Various forms of living radical polymerization have been proposed with dormant chains.
  • the ATRP method atom transfer radical polymerization method using an alkyl halide as a dormant
  • the RAFT method reversible addition fragmentation chain transfer
  • the NMP method nitroxide mediated polymerization
  • the RAFT method is a method in which a chain transfer agent with a high chain transfer constant called a RAFT agent is added to a normal radical polymerization system to polymerize vinyl monomers.
  • a thioester can be used as a RAFT agent.
  • the amount of RAFT agent can be appropriately selected depending on the molecular weight of the target copolymer. That is, since the RAFT agent is bound to the end of each copolymer, for example, when a 100-mer copolymer is the target product, 0.1 to 3 mol% is used with respect to 100 mol% of the monomer. do it.
  • the radical polymerization initiator used for RAFT polymerization is not particularly limited, and may be appropriately selected from known initiators such as azo compounds, peroxides, and redox initiators.
  • azo compounds examples include 2,2'-azobisisobutyronitrile (AIBN), 2,2'-azobis 2,4-dimethylvaleronitrile, 2,2'-azobis(2-methylpropionamidine) dihydrochloride salts, 4,4'-azobis(4-cyanovaleric acid).
  • AIBN 2,2'-azobisisobutyronitrile
  • 2,2'-azobis 2,4-dimethylvaleronitrile 2,2'-azobis(2-methylpropionamidine) dihydrochloride salts
  • 4,4'-azobis(4-cyanovaleric acid 4,4'-azobis(4-cyanovaleric acid
  • the polymerization initiator is generally preferably 0.1 to 50 mol % with respect to 1 mol of the RAFT agent.
  • the reaction in the RAFT method is generally carried out at 40°C to 150°C, depending on the radical polymerization initiator used. Polymerization is often carried out under atmospheric pressure, but polymerization is also possible under pressure.
  • the RAFT method can be performed in the absence of solvent, but it can also be performed in the presence of solvent.
  • Solvents used as necessary are not particularly limited, and known solvents can be used.
  • the reaction can be carried out even in water, and the reaction proceeds even in emulsion polymerization.
  • emulsifier used at that time nonionic emulsifiers, cationic emulsifiers, and anionic emulsifiers that can be used in general emulsion polymerization can be used.
  • Step (2) A step of reacting the obtained copolymer (precursor) with a precursor compound represented by Formula 4' to obtain a specific copolymer.
  • Z is a group containing a click reaction site (eg, cyclic alkyne), and is preferably a group selected from the groups represented by Formula C already described.
  • * is the bonding position with L10.
  • L 10 represents a divalent group, has the same definition as L 2 in Formula 2, and the same applies to its preferred form.
  • the hydroxy group of the copolymer (precursor) and the carboxyl group of the precursor compound represented by Formula 4′ form an ester bond (condensation) to synthesize the desired specific copolymer. be done.
  • the method for forming an ester bond is not particularly limited, for example, a copolymer (precursor) and a precursor compound are heated in the presence of a condensing agent, a catalyst, and a solvent at 0 to 150°C (preferably 0 to 100° C.) for 30 minutes to 24 hours (preferably 3 to 15 hours).
  • Condensing agents include triphenylphosphite, N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-carbonyldiimidazole, dimethoxy-1,3, 5-triazinylmethylmorpholinium, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, O-(benzotriazol-1-yl) -N,N,N',N'-tetramethyluronium hexafluorophosphate, and diphenyl (2,3-dihydro-2-thioxo-3-benzoxazolyl)phosphonate can be used, Among them, N,N'-dicyclohexylcarbodiimide is preferred. In this case, N,N-dimethyl-4-amino
  • the specific copolymer Since the specific copolymer has an LCST and a click reaction site, it specifically and irreversibly binds, for example, a compound having an azide group dispersed or dissolved in a liquid medium containing water, form a complex. When this solution is heated to the LCST or above, the complex rapidly aggregates and precipitates and can be easily separated from the liquid medium. For example, if a complex of a compound having an azide group and an antibody is prepared in advance and a conjugate with a specific copolymer is prepared, or a compound having an azide group, an antibody, and a specific copolymer are mixed. If a conjugate is prepared by the method, the antigen can be recovered together with the conjugate by binding and complexing the corresponding antigen, followed by heating to the LCST or higher.
  • An antibody-copolymer conjugate according to an embodiment of the present invention is an antibody-copolymer conjugate in which the previously described copolymer and an antibody are bound via a linker represented by the following formula 6: be.
  • L 6 represents a divalent hydrocarbon group which may have a heteroatom, a (poly)oxyalkylene group (the number of carbon atoms in the alkylene group is preferably 1 to 6), and the number of carbon atoms is preferably a straight-chain, branched-chain or cyclic hydrocarbon group having 1 to 20, more preferably a polyoxyalkylene group.
  • the number of repetitions of the polyoxyalkylene group is preferably 2-10, more preferably 2-8.
  • Linkers represented by Formula 6 have N-hydroxysuccinimidyl esters (NHS esters) and can form amide bonds with primary amines (eg, lysine residues) of proteins. That is, it binds to —NH 2 possessed by the antibody to form a complex.
  • the linker has an azide group and bonds with the alkynylene group (click reaction site) of the specific copolymer to form a triazole ring.
  • a preferred form of the antibody-copolymer conjugate includes an antibody-copolymer conjugate having a repeating unit represented by Formula 1 below and a repeating unit represented by Formula 7 below.
  • X 2 represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, and has the same meaning as X 2 in Formula 2, and the preferred forms are also the same.
  • L 2 represents a divalent group, has the same definition as L 2 in formula 2, and the same applies to preferred forms.
  • L6 represents a divalent hydrocarbon group which may have a heteroatom, has the same definition as L6 in formula 6 , and the same applies to the preferred forms.
  • Ab represents an antibody residue. That is, it represents a state in which an amide bond is formed by the primary amine of the antibody and the NHS ester of the linker, and the antibody is fixed to the specific copolymer via the linker.
  • the antibody and the unit 7 are bound in a one-to-one ratio, but the antibody-copolymer conjugate is not limited to the above form, and the multiple primary amines of the antibody are each A state in which the alkynylene group of the specific copolymer is bound via a linker, that is, a crosslinked structure-like structure centered on the antibody molecule may be formed.
  • the antibody-copolymer conjugate may contain a unit 2 represented by formula 2 in addition to the above repeating units. That is, unreacted units 2 may be included.
  • a unit 2 represented by formula 2 in addition to the above repeating units. That is, unreacted units 2 may be included.
  • the molecule of the specific copolymer is smaller than the molecule of the antibody, it may be difficult for many antibodies to bind to the specific copolymer. In this case, unreacted units 2 may remain in the antibody-copolymer conjugate.
  • the ratio of the antibody and the specific copolymer is not particularly limited, but as one form, it is preferably 0.1 to 50 mol of the copolymer per 1 mol of the antibody.
  • the antibody-copolymer conjugate may contain other units (unit 5), etc., which the specific copolymer may contain, and the preferred range of the content of each unit is It is the same as the preferred range.
  • Antibodies are not particularly limited, and known antibodies can be used. Among them, anti-SARS-CoV-2 antibodies are preferable, for example, anti-SARS-CoV-2 (COVID19) nucleocapsid (protein) antibodies and anti-SARS - CoV-2 spike (protein) antibody and the like are more preferred. At this time, the ratio of the content of the specific copolymer to the content of the antibody in the antibody-copolymer conjugate on a molar basis is not particularly limited, but is preferably 0.5-30.
  • a method for concentrating an antigen according to an embodiment of the present invention comprises preparing a mixture A containing the antibody-specific copolymer conjugate and an antigen, and obtaining a complex of the antibody-specific copolymer conjugate and the antigen. and heating the mixture A to 20 to 40° C. to precipitate the complex.
  • Mixture A is, for example, an antibody-specific It can be prepared by adding a copolymer conjugate.
  • the mixture A may contain the antibody-specific copolymer conjugate and the antigen, and the antibody-copolymer conjugate may be prepared in advance and the antigen may be added thereto, or the specific copolymer may be added.
  • An antigen may be added to a solution in which a polymer, a linker, and an antibody are mixed, or a specific copolymer, a linker, an antibody, and an antigen may be mixed at once.
  • the amount of the antibody-specific copolymer conjugate added to the sample is not particularly limited, but there is a correlation between the amount added and the concentration rate. is preferably 0.1 mg or more, more preferably 0.5 mg or more, and even more preferably 1.0 mg or more. Although the upper limit is not particularly limited, generally 5.0 mg or less is preferable. More specifically, the specific copolymer is preferably added in an amount of 0.1 to 10.0 mg per 1 mL of sample fluid (including saliva, etc.).
  • a free specific copolymer (not conjugated with an antibody) may be further added to the mixed solution A.
  • the amount of the specific copolymer to be added is not particularly limited, but is preferably 0.1 to 20 times the molar content of the copolymer contained in the antibody-copolymer conjugate.
  • the method for concentrating the antigen comprises binding the already-described linker and the antibody via an amide bond derived from the amino group of the antibody to prepare an antibody-linker conjugate; And, by preparing a mixed solution B containing a specific copolymer, and in the mixed solution B, the azide group of the antibody-linker complex and the alkynylene group of the copolymer are reacted to bind and forming an antibody-specific copolymer conjugate.
  • Antigens that can be concentrated by this method are not particularly limited, and can be appropriately selected according to the type of antibody possessed by the antibody-copolymer conjugate.
  • the antigen is preferably SARS-CoV-2 protein, more preferably SARS-CoV-2 spike protein, SARS-CoV-2 nucleocapsid protein, and the like.
  • An antibody-specific copolymer conjugate preparation kit comprises the previously described copolymer and a linker.
  • the above kit may contain the specific copolymer and the linker, and may further contain a solvent and the like. Alternatively, it may be added in advance to the developing solution of the antigen test kit by immunochromatography. In this case, the specific copolymer and the linker may be pre-bonded in the developing solution (solution).
  • An antigen test kit is an antigen test kit containing the already-described specific copolymer and a linker.
  • the present antigen test kit may contain a known antigen test device.
  • the specific configuration of the antigen test device is not particularly limited, it is preferably used for testing by an immunological technique.
  • it may be a test strip that is generally used for inspection by immunochromatography, or it may be a device having another configuration.
  • the specific configuration of the antigen test kit is not particularly limited.
  • the specific copolymer, the linker, and the antigen test device may be separated, or the specific copolymer, the linker, and the antigen test device may be integrated.
  • HIPAAm Hydrophilicity IsoPropyl AcrylAmide
  • D,L-2-amino-1-propanol (0.15 mol) and triethylamine (0.15 mol) were thoroughly dissolved in anhydrous chloroform, stirred at 5°C for 20 minutes, and then acryloyl chloride (0.15 mol) was added slowly. and stirred at 5°C for 2 hours.
  • Azido-PEG4-NHS ester (Azido-ethylene glycol (EG4)-NHS ester, Tokyo Kasei Kogyo) powder was dissolved in DMSO (10 mg/ml) and carbonate buffer (PH 8.6) was used to extract different concentrations of azide.
  • -PEG4-NHS ester was prepared. ⁇ -PEG4-NHS ⁇ (anti-COVID-19monoclonal antibody ⁇ >95% ⁇ MyBioSource ⁇ anti-COVID-19 antibody :: anti-Viral COVID 19 Nucleocapsid (NP) Humanized Coronavirus Monoclonal Antibody) ⁇ After the addition, the mixture was stirred at 4°C for 5 hours to introduce an azide group into the antibody, as shown in the following formula.
  • the fluorescence of the antibody sample was measured using the "Infinite" 200 PRO plate reader as follows. Azide-modified antibody samples were diluted to 0.5 mg/ml and applied to 96-microwell plates containing different concentrations of DL-2-aminobutyric acid (50 ⁇ L/well, standard) followed by 5 ⁇ L of fluoresca per well. Min (50 mg/ml) was added, incubated for 15 min in the dark and fluorescence was measured (395 nm/495 nm).
  • the concentration of the copolymer (I) is such that the ratio of the antibody to the copolymer (I) is 1:1, 1:2, 1:4, 1:8, 1:15, 1:30 (all on a molar basis).
  • adjusted to be Successful conjugates were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) compared to antibody and copolymer (I), respectively, used alone.
  • COVID-19 antibody - evaluation of concentration effect of temperature-responsive copolymer The concentration effect of previously prepared COVID-19 antibody-copolymer conjugate (1:1) was evaluated by thermal precipitation method. Purified COVID-19-copolymer conjugate (at a concentration of 1.3 mg/mL and containing 15 equivalents of free specific copolymer in 100 ⁇ L of PBS solution) was added to the microtube and incubated at 37° C. for 5 minutes. Centrifuged (13000 ⁇ g). After that, the supernatant (90 ⁇ L) and the precipitate (10 ⁇ L) were recovered, and the absorbance at 280 nm was measured with an ultraviolet-visible spectrophotometer. The ratio of COVID-19 antibody on a molar basis in liquid and solid phase was estimated and the concentration factor was calculated.
  • COVID-19 antibody-copolymer conjugates (different concentrations, 50 ⁇ L PBS) were mixed with COVID-19 recombinant protein (1.0 mg/mL, 50 ⁇ L PBS) and incubated for 1 hour before adding free polymer to the solution. , and centrifuged at 37° C., 13000 ⁇ g for 5 minutes in a microtube. The supernatant (90 ⁇ L) and the precipitate (10 ⁇ L) were collected, the COVID-19 recombinant protein in the supernatant was measured using a modified lowery protein assay kit according to the manufacturer's instructions, and finally the concentration rate was calculated.
  • COVID-19 recombinant protein 50, 100 pg/mL, 100 ⁇ L PBS
  • COVID-19 antibody-specific copolymer conjugate 1.3 mg/mL, 100 ⁇ L PBS
  • 15 equivalents of free polymer [copolymer (I)] was added and centrifuged at 37° C., 13000 ⁇ g for 5 minutes in a microtube.
  • Supernatant (180 ⁇ L) and precipitate (20 ⁇ L) were collected and the concentrated portion was measured by LFIA.
  • HIPAAm was successfully prepared with two major secondary products. As shown in Figure 1, column chromatography was used to purify HIPAAm, and finally 1 H NMR results were used to confirm the synthesis of HIPAAm (Figure 2).
  • FIG. 5 is the LCST measurement results of P(NIPAAm-co-HIPAAm)
  • FIG. 6 is the copolymer (I)
  • FIG. 7 is the antibody-copolymer (I) conjugate.
  • the measurement was performed using PBS (pH 7.4) as a solvent, a copolymer concentration of 2.0 mg/mL, a heating rate of 0.2° C./min, and a wavelength of 450 nm.
  • LCST was defined as the temperature at which the light transmittance was 50%. From this, it was found that the LCST changed from 37.4°C for P(NIPAAm-co-HIPAAm) to 30.1°C for copolymer (I).
  • anti-COVID-19 monoclonal antibody instead of anti-COVID-19 monoclonal antibody (>95%, MyBioSource, anti-COVID-19 antibody:: anti-Viral COVID 19 Nucleocapsid (NP) Humanized Coronavirus Monoclonal Antibody-, 2 Spike Protein (S1-NTD) Antibody #56996" (Cell Signaling Technology) was attempted to synthesize an azide-COVID-19 antibody in the same manner, and was also successfully synthesized.
  • FIG. 14 shows the results. As the amount of azide (EG) 4-NHS added increased, the amount of azide group bound gradually increased and the amount of amine group bound gradually decreased.
  • EG azide
  • FIG. 9 is a gel image of the COVID-19 antibody-copolymer (I) conjugate by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE).
  • (1) and (11) are protein ladders
  • (2) is antibody: copolymer (I) 1:1 (mol/mol)
  • (3) is 1:2
  • (4) is 1:4
  • (5) is 1:8,
  • (6) is 1:15, (7) is 1:30
  • (8) is copolymer (I) itself
  • (9) is azide-antibody Yes
  • (10) is an antibody.
  • the minimum detection limit for COVID-19 recombinant protein using LFIA is about 50 pg/mL (a5), and 100 pg/mL (a4) shows a weak faint band in the test zone.
  • FIGS. 11(a1), (a2) and (a3) strong positive bands were observed at concentrations of 200 to 1000 pg/mL.
  • Figure 12 is a comparison before and after concentration, (b1) 50 pg/mL without concentration, (b2) 50 pg/mL after concentration, (b3) 100 pg/mL without concentration, (b4) 100 pg/mL after concentration. be. Concentration with the COVID-19 antibody-copolymer conjugate was found to show strong positive bands (indicated by triangles in the figure).
  • FIG. 13 shows the LCST measurement results of the synthesized copolymer (II). From FIG. 13, it was found that the LCST was 29.1°C. From the above results, when the total repeating units are 100 mol%, the content of the SAKIPAAm unit having a click reaction site (corresponding to the unit represented by general formula 2) is 5 to 30 mol% or less. It was found that coalescence (II) allowed antigen concentration at lower temperatures than copolymer (I).
  • this copolymer can dramatically reduce the false positive rate caused by a small amount of target substances (antigens, etc.) in the specimen, and can improve the accuracy of the test. can.
  • target substances antigens, etc.
  • the use of antibody-copolymer conjugates to which SARS-CoV-2 antibodies are immobilized can dramatically increase the accuracy of testing for SARS-CoV-2.

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Abstract

Un copolymère selon la présente invention comprend une unité 1 qui est une unité de répétition représentée par la formule 1 et une unité 2 qui est une unité de répétition représentée par la formule 2 et peut être utilisé pour concentrer un antigène par immobilisation d'un anticorps.
PCT/JP2022/009632 2021-04-12 2022-03-07 Copolymère, kit de préparation de conjugué anticorps-copolymère, conjugué anticorps-copolymère, procédé de concentration d'antigène et procédé de détection d'antigène WO2022219967A1 (fr)

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JP2023514514A JPWO2022219967A1 (fr) 2021-04-12 2022-03-07
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WO2024075559A1 (fr) * 2022-10-06 2024-04-11 国立研究開発法人物質・材料研究機構 Procédé d'extraction d'acide nucléique, procédé d'amplification d'acide nucléique, kit d'extraction d'acide nucléique et kit de test de pcr

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WO2016068271A1 (fr) * 2014-10-31 2016-05-06 日産化学工業株式会社 Composition photosensible, substrat à motif, support de culture cellulaire, et procédé de production de cellules cultivées
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US20170304420A1 (en) * 2014-10-10 2017-10-26 Oxford University Innovation Limited Polymer adjuvant
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WO2024075559A1 (fr) * 2022-10-06 2024-04-11 国立研究開発法人物質・材料研究機構 Procédé d'extraction d'acide nucléique, procédé d'amplification d'acide nucléique, kit d'extraction d'acide nucléique et kit de test de pcr

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